How to prepare for the ‘Free testosterone’ test: rules for taking it
Free testosterone is a small fraction of the hormone that is not bound to blood proteins and can enter cells directly. A test for it is ordered when total testosterone does not give an unambiguous answer. But this is exactly where there is the most confusion: there are several measurement methods, and the quality of preparation affects the result no less than in the case of the total hormone. The editorial team explains how to take this test correctly.
What free testosterone is and when it is measured
In the blood, testosterone circulates in three forms. Most is tightly bound to sex hormone-binding globulin (SHBG), a significant part is loosely bound to albumin, and only about 1–3% is in a free state. The free and albumin-bound fractions together are often called ‘bioavailable’ testosterone.
The logic of the test is simple: if a person has an altered SHBG level, total testosterone can be misleading. High SHBG (for example, in older men or in hyperthyroidism) ‘hides’ a deficiency behind a normal total figure, while low SHBG in obesity or insulin resistance creates the impression of a deficiency where the free fraction is normal.
The Endocrine Society clinical guideline (Bhasin et al., 2018) recommends measuring free testosterone in men with total testosterone near the lower limit of normal and in conditions that alter SHBG concentration. A similar approach was followed by the authors of the European Male Ageing Study (EMAS), where both total and free hormone were assessed together with symptoms to diagnose late-onset hypogonadism.
In women, free testosterone or the calculated ‘free androgen index’ is used when examining for hyperandrogenism, in particular polycystic ovary syndrome. There the hormone concentrations are low, so the requirements for method accuracy are even higher.
Methods: calculation, dialysis and direct immunoassay
The ‘gold standard’ is considered to be equilibrium dialysis: a serum sample is separated by a semipermeable membrane, and the amount of hormone that passed through it is measured. The method is accurate but labour-intensive and expensive, so it is available mainly in reference laboratories.
In everyday practice, calculated free testosterone is used most often. For this, the laboratory measures total testosterone, SHBG and (where possible) albumin, and then computes the free fraction using the formula proposed by Vermeulen and colleagues (1999). The authors showed that the calculation agrees well with equilibrium dialysis.
The third option is the so-called direct, or analogue, immunoassay of free testosterone. The Endocrine Society position statement (Rosner et al., 2007) rated this method as unreliable: its results depend on SHBG concentration and systematically differ from dialysis. That is why the editorial team advises checking with the laboratory which method is used to measure the indicator.
| Method | What needs to be measured | Reliability | Comment |
|---|---|---|---|
| Equilibrium dialysis | The free fraction directly | High | Reference method, not available everywhere |
| Calculation (Vermeulen) | Total testosterone, SHBG, albumin | Good provided total testosterone is accurate | The most common clinical approach |
| Direct analogue immunoassay | The free fraction ‘directly’ | Low | Not recommended by the Endocrine Society position statement |
| Free androgen index | Total testosterone and SHBG | Limited in men | Used more often in women |
From this follows a practical conclusion: the quality of calculated free testosterone cannot be higher than the quality of measuring total testosterone and SHBG. Therefore the rules for preparing for the ‘total’ test fully apply to the ‘free’ one as well.
Basic preparation rules
Since the free fraction is derived from the total hormone, the same circadian rhythm applies to it. Blood is taken in the morning, roughly before 10:00–11:00, at the same time for all repeat tests. Comparing a morning result with an evening one may create the illusion of sharp changes.
Fasting is a mandatory condition. Glucose intake lowers testosterone concentration, as demonstrated in clinical studies, so the last meal should be 8–12 hours before the draw. Water may be drunk; sweet drinks, coffee with sugar or milk — no.
- morning draw, at a time that is stable for you;
- 8–12 hours without food;
- usual sleep the night before, without night shifts;
- no intense training or alcohol in the 24 hours before the test;
- do not take it during an acute illness;
- inform the doctor about all medications, hormones and supplements, including biotin.
Have total testosterone and SHBG done from the same tube and in the same laboratory. If you are offered a ‘calculation’ of the free hormone from results from different days or different laboratories, the resulting figure will make little sense.
Biotin in high doses, often contained in cosmetic vitamin complexes, can affect immunoassays of both testosterone and SHBG. The FDA has warned about such interference. Check with the laboratory how long before the test you should skip intake.
What SHBG and albumin change
SHBG concentration is raised by age, hyperthyroidism, liver disease, oral estrogens and some anticonvulsants, as well as a pronounced calorie deficit. It is lowered by obesity, insulin resistance and type 2 diabetes, hypothyroidism, and the intake of androgens and anabolic steroids, glucocorticoids.
These factors are not ‘preparation errors’ — they reflect the body's real state. But they must be known in order to interpret the result correctly. For example, in a person on a strict diet SHBG may temporarily rise, and free testosterone will be lower than with normal nutrition.
Albumin decreases in severe liver and kidney diseases, with significant protein loss or exhaustion. Most laboratories use a standard albumin value in the calculation, but with pronounced deviations the doctor may ask to measure it separately.
For people engaged in strength sports, it is important to honestly inform the doctor about the use of any hormonal drugs. Exogenous androgens suppress SHBG and the body's own secretion, so without this information the result will be interpreted incorrectly.
Common mistakes and repeat testing
The most common mistake is to have ‘free testosterone’ done by direct immunoassay without the total hormone and SHBG and to draw conclusions from a single indicator. Such a result is difficult to compare with clinical studies that used dialysis or calculation.
The second mistake is a blood draw after breakfast or in the afternoon. The third is a test right after illness, a sleepless night or competition. All these circumstances temporarily lower the hormone and can become a reason for unnecessary treatment.
The Endocrine Society guideline advises confirming low testosterone with a repeat test. The same logic applies to the free hormone: a repeat draw under similar conditions, preferably in the same laboratory, with the same calculation method.
Finally, the units of measurement. Free testosterone is expressed in pmol/L, nmol/L, pg/mL or ng/dL depending on the laboratory. The reference interval is always tied to the method, so rely on the norm given on your form.
Editorial conclusions
Free testosterone is a useful indicator when the total hormone is at the borderline of normal or when SHBG is altered. It is most reliably obtained by equilibrium dialysis or by calculation from total testosterone, SHBG and albumin.
Preparation is the same as for total testosterone: morning, fasting, usual sleep, without training or alcohol the day before, with the doctor informed of all drugs. Direct analogue tests are better not used as a basis for decisions.
We also advise reading our articles on preparing for the total testosterone, SHBG and estradiol tests — these indicators are most often assessed together.
References
- Vermeulen A, Verdonck L, Kaufman JM. A critical evaluation of simple methods for the estimation of free testosterone in serum. J Clin Endocrinol Metab. 1999;84(10):3666–3672.
- Rosner W, Auchus RJ, Azziz R, et al. Position statement: utility, limitations, and pitfalls in measuring testosterone: an Endocrine Society position statement. J Clin Endocrinol Metab. 2007;92(2):405–413.
- Bhasin S, Brito JP, Cunningham GR, et al. Testosterone therapy in men with hypogonadism: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2018;103(5):1715–1744.
- Wu FC, Tajar A, Beynon JM, et al. Identification of late-onset hypogonadism in middle-aged and elderly men. N Engl J Med. 2010;363(2):123–135.
- Brambilla DJ, Matsumoto AM, Araujo AB, McKinlay JB. The effect of diurnal variation on clinical measurement of serum testosterone and other sex hormone levels in men. J Clin Endocrinol Metab. 2009;94(3):907–913.
- U.S. Food and Drug Administration. The FDA warns that biotin may interfere with lab tests: FDA safety communication. 2017.
Andriy Melnyk
A strength-sports coach and author of programs for beginner and intermediate levels. Writes about training planning.
